This project focuses on glycoconjugates of Schwann cells and oligodendrocytes during myelination and demyelination. A major aspect of the research concerns the myelin-associated glycoprotein (MAG), which is localized in periaxonal glial membranes of myelinated fibers and functions in transmitting signals between axons and myelin-forming cells. MAG is in the "siglec" subgroup of the immunoglobulin superfamily and binds to glycoconjugates containing terminal alpha2-3-linked sialic acid, suggesting that its axonal receptor or ligand could be a glycoprotein or ganglioside. Previous studies from our laboratory and others on MAG-null mice indicate that the most important functions of MAG are different in the PNS and CNS. In the PNS, MAG is essential for signaling from Schwann cells to axons that is needed for the normal maintenance of myelinated axons. In the absence of MAG, the pathology in the PNS is characterized by degeneration of myelinated axons. This is preceded by a reduction of axonal caliber caused by cytoskeletal abnormalities including decreased expression and phosphorylation of neurofilaments, due in part to decreased activities of extracellular signal regulated kinases 1 & 2 (ERK 1/2) and cyclin dependent kinase 5 (cdk5). In vitro experimental paradigms of MAG interaction with neurons demonstrate that the presence of MAG causes elevated ERK 1/2 and cdk5 activities and increased expression of phosphorylated neurofilaments and other cytoskeletal elements. Pharmacological experiments completed this year demonstrate that activation of cytoskeleton-associated ERK 1/2 occurs through the traditional Ras-Raf-Mek pathway and is negatively regulated by protein kinase A. The well-known role of MAG as one of several white matter inhibitors of neuronal regeneration also shows that MAG is able to influence the properties of axons, but it is unclear how this capacity to inhibit neurite outgrowth relates to its normal function in glia-axon interactions within the periaxonal region of myelinated axons. The capacity to inhibit outgrowth of plastic regenerating neurites may be an early manifestation of a MAG-mediated signaling system that promotes axonal maturation to eventually optimize their structure for rapid conduction of action potentials in mature myelinated axons. In the past few years, research in many laboratories on the inhibition of neurite outgrowth has provided much new information about a neuronal receptor for MAG, which appears to involve a complex of the Nogo receptor, the p75 neurotrophin receptor and gangliosides. A principal goal of our ongoing research is to determine how these findings with immature plastic neurons relate to MAG-mediated signaling within the periaxonal compartment of myelinated axons that is essential for their normal maintenance. Our recent experiments described above on ERK 1/2 activation in cultured neurons indicated that this effect of MAG signaling does involve the Nogo receptor. In addition, we showed by western blotting that the Nogo receptor and the p75 neurotrophin receptor are present in a membrane fraction isolated from white matter that is enriched in axolemma and periaxonal oligodendroglial membranes. Furthermore, an interaction of the Nogo receptor with MAG at this location was demonstrated by co-immunoprecipitation. In contrast to the PNS, the most important function of MAG in the CNS appears to be signaling in the opposite direction from axons to oligodendrocytes to promote their differentiation and survival. Morphological studies on the CNS of MAG-null mice revealed a significant delay of myelination, aberrant or redundant myelin loops and abnormal paranodal structures. Thus in the absence of MAG, some oligodendrocytes do not seem to be efficient at determining when, where and how much myelin to form. Furthermore, there is a loss of oligodendroglial proteins and degeneration of periaxonal oligodendroglial processes in aging MAG-null mice consistent with a "dying back" oligodendrogliopathy, similar to that occurring in some demyelinating lesions of multiple sclerosis. These findings suggest that MAG functions as the receptor for an axonal signal that enhances the process of myelination and the vitality of oligodendrocytes. Experiments are in progress with primary oligodendrocyte cultures and oligodendroglial cell lines to demonstrate and characterize a MAG-mediated signaling pathway that promotes the differentiation and/or survival of these cells. Two approaches, which we are exploring to activate MAG-mediated signaling in cultured oligodendrocytes, are cross linking with anti-MAG antibodies and growth on a substratum containing a 2,3-sialylated glycoprotein. Antibody cross-linking of MAG leads to its redistribution into detergent insoluble, raft-like complexes, increased phosphorylation of Fyn tyrosine kinase, dephosphorylation of serine and threonine residues in some other proteins, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. These in vitro results support the hypothesis MAG functions in axon-glia interactions to activate a signaling cascade affecting the properties of oligodendrocytes.